DESCRIPTION: This research team has recently created a mouse that is heterozygous for a null mutation in the DNA repair gene XRCC1 (X-ray repair cross-complementing). The presence of this and other members of the XRCC class of DNA repair genes protects cells against the lethal effects of ionizing radiation and alkylating agents. The XRCC1-complemented CHO cell line, EM9, is hypersensitive to many mutagenic agents, and demonstrates a 10-fold increase in baseline levels of sister chromatid exchange over wild type cells. In addition, EM9 cells are deficient in rejoining DNA single-strand breaks. Homozygous knockout embryos generated by intercrossing mice heterozygous for a novel, null XRCC1 mutation die between embryonic day (E) 7.5 and 9.5. Therefore, XRCC1 may be essential for embryonic development in the mouse. As such, this mutation acts at the earliest time of any known mammalian DNA repair deficiencies. This proposal is to further characterize the XRCC1 knockout mutants and to determine the developmentally essential role of XRCC1 in repairing DNA damage during mammalian embryogenesis. Because of the important role XRCC1 plays in modulating the damaging effects of various mutagens, the characterization of the knockout mice and cells derived from them will improve understanding of the mechanisms employed by cells to protect against DNA damage, particularly DNA base damage induced by exposure to radiation and radio-mimetic chemicals. Accordingly, specific aim 1 will be to determine the cellular basis for the lineage-specific abnormal phenotype of the XRCC1 null mutant embryo. The hypothesis underlying this aim is that the lethal null mutant phenotype results from the accumulation of spontaneous damage, which is occurring in each cell generation, to critical threshold levels during early embryogenesis. The principal investigator predicts that both spontaneous base damage and its repair by XRCC1 are ubiquitous but that the embryonic and extraembryonic lineages differ in their tolerance for unrepaired damage. The experimental approach to test this prediction will involve some descriptive observations and also the use of chimeric arrangements with normal embryonic cells to look for the possibility of rescue and further development of XRCC1 -/- embryonic cells along embryonic cell lineages as well as the extraembryonic cell lineages. Specific aim 2 will be to analyze the molecular mechanisms of the response to DNA damage in XRCC1-deficient cells. The hypothesis underlying this aim is that p53 mediates the molecular responses to DNA damage, including apoptosis, in XRCC1 null mutant embryos. This hypothesis predicts that the p53-/- null mutation will rescue XRCC1 null mutant embryos to a later stage of development and diminish their apoptotic phenotype. The experimental approach to test this hypothesis will involve interbreeding XRCC1 and p53 mutants. Specific aim 3 will be to analyze the genetic consequences of the XRCC1 null mutant phenotype in vitro and in vivo. The hypothesis underlying this aim is that XRCC1 function is essential for DNA strand break repair in all cell types and developmental stages owing to its role in base pair excision repair. This hypothesis predicts that the homozygous null mutants will exhibit elevated mutation rates, genetic instability and unrepaired chromosomal damage and that developing individuals would have impaired meiosis as a result of germ cell-specific XRCC1 dysfunction. The experimental approach to test this hypothesis will involve generating a conditional knockout for XRCC1 using Cre/lox technology. An overall rationale for the planned studies is that understanding the role of DNA repair genes during natural development, when it appears that spontaneous breaks do occur and that failure to repair them is embryo-lethal, could establish an essential role for these DNA repair genes during development and that there is a heretofore unknown, functionally relevant, incidence of DNA strand breaks that occurs during normal development. In addition, identifying tissue-specific and/or stage-specific role(s) for XRCC1 and similar gene products during development might lead to the identification of functionally significant polymorphisms in these genes that increase the risk of carriers to diseases resulting from unrepaired DNA damage.